Method for quantitating cholesterol present in low density lipoproteins

ABSTRACT

A method for quantifying LDL cholesterol, by which LDL cholesterol is separately quantified simply without requiring complicated centrifuge operation is disclosed. The method for quantifying cholesterol in low density lipoprotein according to the present invention comprises a first step of erasing cholesterol in high density lipoprotein, very low density lipoprotein and chylomicron in a test sample, and a second step of quantifying cholesterol remaining in the test sample.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/JP97/03663 which has an Internationalfiling date of Oct. 13, 1997 which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a method for quantifying cholesterol inlow density lipoprotein (LDL, The cholesterol in low density lipoproteinwill be hereinafter also referred to as “LDL cholesterol”. In thepresent specification, the term “cholesterol” includes both ester typecholesterol and free cholesterol), which is important for the diagnosisof arterial sclerosis.

BACKGROUND ART

LDL plays a main role in transportation of cholesterol in blood and mostof the cholesterol deposited on the blood vessel wall in pultaceousarterial sclerosis is originated from LDL. Increase in the amount of LDLin plasma is one of the major risk factors in pultaceous sclerosis suchas ischemic heart disease, so that separate quantification of LDLcholesterol is clinically important.

Conventional methods for quantifying LDL cholesterol include a methodcomprising two steps, that is, a fractionation operation and anoperation for quantifying the cholesterol, and a method in which theblood levels of total cholesterol, HDL cholesterol and triglyceride aredetermined and the amount of the LDL cholesterol is determined accordingto the Friedewald's equation.

Fractionation operation includes ultracentrifugation method,precipitation method, immunochemical method and the like. In theultracentrifugation method, LDL is separated exploiting the differencein the specific gravity by an ultracentrifuge, and the amount of thecholesterol therein is measured. In the precipitation method, anti-HDLantibody, polyanion and a divalent cation are added to form an insolubleprecipitate, and the LDL cholesterol in the supernatant aftercentrifugation is quantified (WPI Acc No.85-116848/20). In theimmunochemical method, anti-HDL antibody, anti-VLDL antibody and anti-CMantibody are bound to latex particles, and the latex particles areremoved by centrifugation or by passing through a filter afteragglutination, followed by quantifying the LDL cholesterol (WPI Acc No.84-301275/49). However, these conventional methods are problematic insimplicity or cost.

According to the Friedewald's equation, the amount of LDL cholesterol iscalculated by subtracting the amount of the HDL cholesterol from theamount of the total cholesterol, and then further subtracting the ⅕ ofthe amount of the triglyceride. However, since this method does not takethe influence by the diet and the individual difference, this method isproblematic in accuracy.

Recently, a method for quantification of LDL cholesterol, which does notrequire fractionation operation, has been reported (WPI Acc No.83-766269/38). However, in this method, the specificity to LDL is notsufficient.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a method forquantifying LDL cholesterol, by which LDL cholesterol is separatelyquantified simply without requiring complicated centrifuge operation.

The present inventors discovered that the amount of cholesterol in LDLcan be quantified by erasing the cholesterol other than the cholesterolin the low density lipoprotein in the first step, and by measuring theremaining cholesterol in the subsequent second step, thereby completingthe present invention.

That is, the present invention provides a method for quantifyingcholesterol in low density lipoprotein in a test sample which maycontain low density lipoprotein, high density lipoprotein, very lowdensity lipoprotein and/or chylomicron, which method comprises a firststep of erasing cholesterol in high density lipoprotein, very lowdensity lipoprotein and chylomicron in a test sample, and a second stepof quantifying cholesterol remaining in said test sample.

By the present invention, a method for quantifying LDL cholesterol, bywhich LDL cholesterol is separately quantified simply without requiringcomplicated centrifuge operation, was provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the correlation between the results of the measurement ofthe LDL cholesterol in Example 1 and the amount calculated by theFriedewald's equation.

FIG. 2 shows the correlation between the results of the measurement ofthe LDL cholesterol in Example 2 and the amount calculated by theFriedewald's equation.

FIG. 3 shows the correlation between the concentration of LDLcholesterol in the absence or presence of HDL, VLDL and CM and theabsorbance measured by the present invention in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

Cholesterol contained in lipoproteins includes ester type cholesterol(cholesterol ester) and free cholesterol. In this specification, theterm “cholesterol” includes both of these unless otherwise specified.

The test sample subjected to the method of the present invention may beany sample which may contain lipoproteins such as HDL, LDL, VLDL and CM.Examples of the test samples include body fluids such as blood, sera andplasma as well as dilutions thereof, although the test samples are notrestricted thereto.

The method of the present invention comprises a first step and a secondstep. In the first step, the cholesterol in HDL, VLDL and CM in the testsample is erased, and in the second step, the cholesterol remaining inthe test sample is quantified. Since the cholesterol in HDL, VLDL and CMis erased in the first step, the cholesterol quantified in the secondstep is mainly the cholesterol in LDL in the test sample.

The term “erase” in the first step herein means to decompose thecholesterol and to make the decomposed products undetectable in thesubsequent second step. The methods for selectively erasing thecholesterol in the lipoproteins other than LDL, that is, in HDL, VLDL,CM and the like include the following methods.

That is, cholesterol esterase and cholesterol oxidase are made to act onthe test sample in the presence of a surfactant which acts onlipoproteins other than low density lipoprotein, and the generatedhydrogen peroxide is erased.

Methods for erasing hydrogen peroxide include a method in which thehydrogen peroxide is decomposed to water and oxygen by catalase; and amethod in which a phenol-based or an aniline-based hydrogen donorcompound is reacted with the hydrogen peroxide to convert the hydrogenperoxide to a colorless quinone, although the methods for removinghydrogen peroxide are not restricted to these methods.

The concentration of the cholesterol esterase in the reaction mixture inthe first step may preferably be about 0.2 to 1.0 U/ml, and thecholesterol esterase may preferably be originated from a bacteriumbelonging to the genus Pseudomonas. The concentration of the cholesteroloxidase may preferably be about 0.1 to 0.7 U/ml, and the cholesteroloxidase may preferably be originated from a bacterium or yeast. Theconcentration of the catalase may preferably be about 40 to 100 U/ml andthe concentration of the peroxidase by which the hydrogen peroxide isconverted to a colorless quinone may preferably be about 0.4 to 1.0U/ml. The concentration of the phenol-based or aniline-based hydrogendonor compound may preferably be about 0.4 to 0.8 mmol/l.

Preferred surfactants which act on the lipoproteins other than LDL,which are used in the first step, include polyalkylene oxide derivativeshaving HLB values of not less than 13 and not more than 15, preferablynot less than 13 and not more than 14. Examples of the derivatives hereinclude condensation products with higher alcohols, condensationproducts with higher fatty acids, condensation products with higherfatty acid amides, condensation products with higher alkylamines,condensation products with higher alkylmercaptane and condensationproducts with alkyl phenols. The method for calculating HLB ofsurfactants is well-known, and is described in, for example, HiroshiHORIUCHI, “New Surfactants”, 1986, Sankyo Shuppan.

Preferred specific examples of the polyalkylene oxide derivatives havingHLB values of not less than 13 and not more than 15 includepolyoxyethylene lauryl ether, polyoxyethylene cetyl ether,polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether,polyoxyethylene octyl phenyl ether, polyoxyethylene nonylphenyl etherand the like, of which HLB value is not less than 13 and not more than15, although the surfactant is not restricted thereto.

As the surfactant used in the first step, a cation surfactant may alsobe used. In this case, as the cation surfactant, those having thequaternary ammonium salt as a hydrophilic group, represented by thefollowing formula (I), are preferred.

wherein R independently represents C₁-C₈ linear alkyl group and R¹represents C₃-C₂₀ alkenyl group.

The concentration of the above-mentioned surfactants used in the firststep may preferably be about 0.1 to 10 g/l, more preferably about 0.5 to5.0 g/l.

The reaction in the first step may preferably be carried out in a bufferwith a pH of 5 to 8, and the buffer may preferably be one containing anamine such as Tris buffer, triethanolamine or Good's buffer. Especially,Bis-Tris, PIPES, MOPSO, BES, HEPES and POPSO which are Good's buffer arepreferred. The concentration of the buffer may preferably be about 10 to500 mM.

To inhibit the reaction with LDL and to increase the degree of erasingof the other lipoproteins, a divalent metal ion may be contained in thereaction mixture. Preferred examples of the divalent metal ion includecopper ion, iron ion and magnesium ion. Among these, magnesium ion isespecially preferred. The concentration of the divalent metal ion maypreferably be about 5 to 200 mM.

A lipoproteinase may optionally be added to the reaction mixture in thefirst step. Addition of this enzyme is preferred because especially thecholesterol in VLDL easily reacts. The concentration of this enzyme inthe reaction mixture may preferably be about 5.0 to 10.0 U/ml.

The reaction temperature in the first step may preferably be about 25°C. to 40° C., and 37° C. is best preferred. The reaction time may beabout 2 to 10 minutes.

In the subsequent second step, the cholesterol remaining in the testsample is quantified. This may be carried out by, for example, adding asurfactant which acts on at least LDL and quantifying the hydrogenperoxide by the action of the cholesterol esterase and the cholesteroloxidase added in the first step. Here, the surfactant which acts on atleast LDL may be a surfactant which selectively acts on LDL alone or maybe a surfactant which acts on all lipoproteins.

Preferred examples of the surfactant which acts on all lipoproteinsinclude polyalkylene oxide derivatives having HLB values of not lessthan 11 and not more than 13, preferably not less than 12 and not morethan 13. Examples of the derivatives here include condensation productswith higher alcohols, condensation products with higher fatty acids,condensation products with higher fatty acid amides, condensationproducts with higher alkylamines, condensation products with higheralkylmercaptane and condensation products with alkyl phenols.

Preferred specific examples of the polyalkylene oxide derivatives havingHLB values of not less than 11 and not more than 13 includepolyoxyethylene lauryl ether, polyoxyethylene cetyl ether,polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether,polyoxyethylene octyl phenyl ether, polyoxyethylene nonylphenyl etherand the like, of which HLB value is not less than 11 and not more than13, although the surfactant is not restricted thereto.

Examples of the surfactants which selectively act on LDL alone includeanion surfactants. Although the anion surfactants used here are notrestricted, those having one or more aromatic rings to which one or moreC₄-C₁₈ linear or branched alkyl groups are attached are preferred. Here,the aromatic ring may preferably be those consisting of carbon atoms andhydrogen atoms, such as benzene, naphthalene and diphenyl. Those inwhich one or more hydrophilic groups such as sulfonate group areattached to the above-mentioned aromatic ring are further preferred.Preferred examples of such anion surfactants include those representedby the following formulae (II) to (VI).

In formulae (II) to (VI), R independently represents C₄-C₁₈ linear orbranched alkyl group. Preferred examples of the anion surfactants whichmay be used in the second step also include higher alcohol sodiumsulfate and the like.

The concentration of the surfactant used in the second step maypreferably be about 0.1 to 100 g/l, more preferably about 1 to 50 g/l.

Other preferred reaction conditions in the second step are the same asthe preferred reaction conditions in the first step.

The present invention will now be described more concretely by way ofexamples thereof. It should be noted, however, the present invention isnot restricted to the examples below.

EXAMPLE 1

First Reagents BES buffer, pH 6.0 100 mmol/l HDAOS:N-(2-hydroxysulfopropyl)-3,5-dimethyoxy- 0.7 mmol/l aniline Cholesterolesterase originated from a bacterium belong- 0.8 U/ml ing to the genusPseudomonas (trademark “CEN” commercially available from Asahi ChemicalIndustry Co. Ltd.) Cholesterol oxidase originated from a bacteriumbelong- 0.5 U/ml ing to the genus Streptomyces (trademark “COO”commercially available from Toyobo Co. Ltd.) Catalase 80 U/ml Magnesiumchloride 10 mmol/l Emulgen B66 commercially available from KAO 0.2%CORPORATION (polyoxyethylene derivative (HLB = 13.2)) Second ReagentsBES buffer, pH 7.0 50 mmol/l 4-aminoantipyrine 4.0 mmol/l Peroxidase 2.4U/ml Sodium azide 0.1% Emulgen A60 commercially available from KAO 5.0%CORPORATION (polyoxyethylene derivative (HLB = 12.8))

To each of 4 samples having a volume of 4 μl containing purified HDL,LDL, VLDL and CM at a concentration of 100 mg/dl in terms ofcholesterol, respectively, 300 μl of the above-described first reagentswhich had been preliminarily warmed at 37° C. were added and each of theresulting mixtures was allowed to react at 37° C. for 5 minutes.Thereafter, 100 μl of the second reagents were added to each mixture andeach of the resultants was allowed to react for 5 minutes, followed bymeasurement of absorbance of each reaction mixture at 600 nm. Based onthe measured absorbances, the amounts of cholesterol were calculated andthe ratio of the thus calculated amount to the amount of the cholesterolin the sample was calculated, which is defined as capture ratio. Theresults are shown in Table 1 below.

TABLE 1 Capture Ratio CM VLDL LDL HDL <1.0% <5.0% 70.0% <1.0%

As shown in Table 2, by the above-described method, most of thecholesterol in LDL was captured while the cholesterol in otherlipoproteins was not substantially captured, so that the cholesterol inLDL can be selectively quantified.

EXAMPLE 2

First Reagents PIPES buffer, pH 7.0 50 mmol/l HDAOS 0.7 mmol/lCholesterol esterase originated from a bacterium belong- 0.8 U/ml ing tothe genus Pseudomonas (trademark “CEN” commercially available from AsahiChemical Industry Co. Ltd.) Cholesterol oxidase originated from abacterium belong- 0.5 U/ml ing to the genus Streptomyces (trademark“COO” commercially available from Toyobo Co. Ltd.) Catalase 80 U/mlMagnesium chloride 10 mmol/l Emulgen B66 commercially available from KAO0.2% CORPORATION Second Reagents PIPES buffer, pH 7.0 50 mmol/l4-aminoantipyrine 4.0 mmol/l Peroxidase 2.4 U/ml Sodium azide 0.1%Triton X100 3.0%

The same procedures as in Example 1 were repeated and the reactivitywith each lipoprotein was measured. The results are shown in Table 2below.

TABLE 2 Capture Ratio CM VLDL LDL HDL <1.0% <5.0% 71.0% <1.0%

EXAMPLE 3

Using sera of normal persons as test samples, the procedures in Examples1 or 2 were repeated to measure the concentrations of LDL cholesterol.As controls, the concentrations of LDL cholesterol in the sera weremeasured employing the Friedewald's equation (CLIN. CHEM., 41, 1414,1995). The results are shown in FIGS. 1 and 2 showing the correlationtherebetween.

As shown in FIGS. 1 and 2, the results of measurement by the bothmethods well agreed, so that it was proved that the cholesterol in LDLcan be quantified accurately by the method of the present invention.

EXAMPLE 4

First Reagents Good's buffer, pH 7.0 50 mmol/l HDAOS 0.7 mmol/lCholesterol esterase 0.8 U/ml Cholesterol oxidase 0.5 U/ml Catalase 80U/ml Cation surfactant (lauryl trimethylammonium chloride) 0.1% SecondReagents 4-aminoantipyrine 4.0 mmol/l Peroxidase 2.4 U/ml Sodium azide0.1% Nonionic surfactant (polyoxyethylene lauryl ether) 0.1% (Thenonionic surfactant was used in the second reaction.)

Twenty microliters of a sample was mixed with 180 μl of the firstreagents preliminarily warmed at 37° C. and the resulting mixture wasallowed to react at 37° C. for 5 minutes. Then 60 μl of the secondreagents were added and the resulting mixture was allowed to react at 5minutes, followed by measurement of the absorbance at 600 nm.

FIG. 3 shows the relationship between the concentration of the LDLcholesterol and the absorbance. As can be seen from FIG. 3, LDLcholesterol can be measured specifically and concentration-dependentlyeven in the presence of HDL, VLDL and CM.

EXAMPLE 5

Using sera as test samples, the same procedures as in Example 4 wererepeated to determine the concentration of the LDL cholesterol. Ascontrols, the concentrations of LDL cholesterol in the sera weremeasured employing the Friedewald's equation (CLIN. CHEM., 41, 1414,1995). The results are shown in Table 3. As shown in Table 3, theresults obtained by the method of the present invention well agreed witthe results calculated according to the Friedewald's equation.

TABLE 3 Friedewald Example 4 Sample 1 73.0 60.1 2 91.0 85.8 3 136.4124.0 4 97.7 98.0 5 75.2 81.8 6 195.7 195.4 7 140.5 112.9 8 112.8 113.29 160.6 153.5 10  120.4 111.1

What is claimed is:
 1. A method for quantifying cholesterol in lowdensity lipoprotein in a test sample which may contain low densitylipoprotein, high density lipoprotein, very low density lipoproteinand/or chylomicron, which method comprises a first step of enzymaticallyerasing cholesterol in high density lipoprotein, very low densitylipoprotein and chylomicron in a test sample, and a second step ofquantifying cholesterol remaining in said test sample, wherein saidfirst step is carried out by the action of cholesterol esterase andcholesterol oxidase in the presence of a surfactant which acts onlipoproteins other than the low density lipoprotein, and by eliminatingthe generated hydrogen peroxide.
 2. The method according to claim 1,wherein said second step comprises adding a surfactant which acts on atleast the low density lipoprotein, and quantifying the hydrogen peroxidegenerated by the actions of said cholesterol esterase and cholesteroloxidase.
 3. The method according to claim 2, wherein said surfactantwhich acts on at least the low density lipoprotein is a surfactant whichacts on all of the lipoproteins.
 4. The method according to any one ofclaims 1 to 3, wherein said surfactant which acts on lipoproteins otherthan the low density lipoprotein, which is used in said first step is apolyalkylene oxide derivative having an HLB value of not less than 13and not more than
 15. 5. The method according to claim 3, wherein saidsurfactant which acts on all of the lipoproteins used in said secondstep is a polyalkylene oxide derivative having an HLB value of not lessthan 11 and less than
 13. 6. The method according to claim 1, whereinsaid surfactant which acts on lipoproteins other than the low densitylipoprotein, which is used in said first step, is a cationic surfactant.7. The method according to claim 6, wherein said cationic surfactant hasa quaternary ammonium salt.
 8. The method according to claim 2, whereinsaid surfactant which acts on at least the low density lipoprotein,which is used in the second step, is an anionic surfactant.
 9. Themethod according to claim 1, wherein said first step is carried out at aconcentration of said surfactant of 0.1 to 10 g/l.
 10. The methodaccording to claim 5 or 8, wherein said polyoxyalkylene derivativehaving an HLB value of not less than 11 and less than 13 or said anionicsurfactant used in said second step has a concentration of 1 to 100 g/l.11. The method according to claim 1, wherein said first and second stepsare carried out in a buffer with a pH of 5 to
 8. 12. The methodaccording to claim 11, wherein said buffer contains an amine.
 13. Themethod according to claim 1, wherein said first and second steps arecarried out at a temperature of 25 to 40° C.